1. Field of the Invention
The present invention is concerned with methods of cloning and expressing the leukotoxin gene from Fusobacterium necrophorum (F. necrophorum), sequencing and characterizing the leukotoxin protein expressed by this gene, truncating the gene into a series of nucleotide sequences, amplifying these sequences, expressing and recovering the polypeptides encoded by the nucleotide sequences, and utilizing the protein and the polypeptides in recombinant vaccines in order to confer effective immunity against infection caused by the production of leukotoxin by F. necrophorum. More particularly, it is concerned with production of an inactivated recombinant leukotoxin vaccine generated by amplifying five leukotoxin gene fragments and one upstream region through PCR, digesting the nucleotide sequences encoded by the gene fragments with restriction enzymes, expressing the polypeptide sequences coded by the nucleotide sequences through an expression vector, recovering these proteins as five truncated leukotoxin proteins (or polypeptides), purifying these proteins (or polypeptides) to apparent homogeneity, with or without inactivation of the truncated and full length proteins, and combining the inactivated recombinant leukotoxins with adjuvants.
2. Description of the Prior Art
Liver abscesses in feed lot cattle are a serious economic problem, causing condemnation of over 3 million livers and an estimated loss of $15 million annually in the United States. This estimate is based primarily on condemnation of liver and other organs, and does not include economic losses stemming from reduced feed intake, reduced feed efficiencies, decreased carcass dressing percentage and lowered weight gains. A number of studies have confirmed that cattle with abscessed livers gain less (average 4-5%) and have reduced feed efficiencies (average 7%) compared with cattle having healthy livers. The average incidence of abscessed liver in grain-fed cattle approximates 25-30%. To a lesser extent, liver abscesses in sheep and goats are also an economic problem.
F. necrophorum is a gram-negative, rod-shaped, nonsporeforming, nonmotile, strictly anaerobic and pleomorphic organism. Morphologically, the organism varies from short rods to filamentous with pointed and rounded ends. Cell lengths range from coccoid bodies of 0.5-0.7 μm in diameter to filaments over 100 μm. Surface colonies are 1-2 mm in diameter, circular, transparent to opaque, and with some strains producing α or β hemolysis. The organism ferments glucose, fructose and maltose only weakly with final pH around 5.0-6.3. It ferments lactate to acetate, propionate, and butyrate. Butyrate is the major product from lactate fermentation. Indole is produced from peptone. F. necrophorum has been isolated from the normal flora in the oral cavity, gastrointestinal cavity, and genitourinary tract of humans and animals. The organism is also known to survive in the soil.
F. necrophorum is a normal inhabitant of the gastrointestinal tracts of animals and humans. Virulence factors and pathogenic mechanisms that contribute to the transition of this otherwise commensal organism to a pathogen are poorly understood. A leukotoxin, endotoxin, hemolysin, hemagglutinin, and several enzymes such as deoxyribonuclease and proteases have been suggested as possible virulence factors. However, several studies implicate leukotoxin, a protein cytotoxic to ruminantpolymorphonuclear cells, as the major virulence factor. The importance of leukotoxin as a virulence factor in F. necrophorum infections is indicated by a correlation between toxin production and ability to induce abscesses in laboratory animals, an inability of nonleukotoxin-producing strains to induce foot abscesses in cattle following intradermal inoculation, and a relationship between antileukotoxin antibody titers and protection against infection in experimental challenge studies.
F. necrophorum is an opportunistic pathogen that is the primary etiologic agent of liver abscesses in ruminant animals. (Scanlan, et al., (1983)Bovine rumenitis-liver abscess complex: a bacteriological review. Cornell Vet. 73:288-297; Nagaraja, T. G. et al., (1998) Liver abscesses in feedlot cattle: A review. J. Anim. Sci., 76:287-298; and Tan, et al., (1996) Fusobacterium necrophorum infections: virulence factors, pathogenic mechanism and control measures. Vet. Res. Comm., 20:113-140). The organism has been recognized as an animal and human pathogen since the late 1800s, and is associated as a primary or secondary etiologic agent with numerous necrotic disease conditions in domestic and wild animals. In addition to liver abscesses, the organism is also the primary etiologic agent of foot rot, foot abscesses, calf diphtheria, and is frequently isolated from cases of mastitis, metritis, and necrotic lesions of the oral cavity.
Liver abscesses in cattle are part of a disease complex where the abscessation is secondary to primary foci of infection in the rumen epithelium. The pathogenesis can be summarized as follows: (1) ruminal lesions are induced by acidosis that follows rapid change in diet from high-roughage to high grain, prolonged feeding of high grain diet, or occasionally by foreign body penetration of the rumen epithelium; (2) bacteria present in the rumen invade the epithelium and form focal abscesses in the rumen wall; and (3) bacteria enter the portal circulation, and are carried to the liver where they localize in the parenchyma with subsequent abscess formation.
The ability of F. necrophorum to establish in the liver is attributed to the production of a toxin which is a secreted protein of high molecular weight active against leukocytes from ruminants called leukotoxin (or leucocidin). The toxin is a soluble extracellular protein that is cytotoxic to neutrophils, macrophages, hepatocytes, and ruminal cells. The leukotoxin protects against phagocytosis and is believed to aid in the establishment of F. necrophorum in the liver by directly impairing the normal defense mechanism and indirectly by the damage caused by cytolytic products released from neutrophils and macrophages to the hepatic cells. Therefore, the leukotoxin elaborated from F. necrophorum plays a critical role in F. necrophorum infection of the liver and is believed to be the primary virulence factor in the pathogenesis of liver abscesses (Tan et al., 1996).
Four biotypes (A, B, AB and C) of F. necrophorum have been described. (Langworth, (1977) Fusobacterium necrophorum: its characteristics and role as an animal pathogen. Bacteriol. Rev. 41:373-390) Biotype A, most frequently isolated from liver abscesses, is more pathogenic than biotype B, which predominates in ruminal wall abscesses. Biotypes AB and C are rarely isolated in liver abcesses (Berg, et al., (1982) Studies of Fusobacterium necrophorum from bovine hepatic abscesses: Biotypes, quantitation, virulence, and antibiotic susceptibility. Am. J. Vet. Res. 43:1580-1586), and biotype A has pathogenicity intermediate that of biotypes A and B while biotype C is non-pathogenic. (Shinjo, et al., (1990) Recognition of biovar C of Fusobacterium necrophorum (flugge) Moore and Holdeman as Fusobacterium pseudonecrophorum sp. nov., nom. rev. (ex prevot 1940) Int. J. Sys. Bacteriol. 41:395-397) Biotypes A and B, the most frequent types encountered in liver abscesses, have been assigned subspecies status: subsp. necrophorum and subsp. funduliforme, respectively (Shinjo et al., 1990). The subsp. necrophorum is more virulent, produces more leukotoxin and hemagglutinin, and is more frequently isolated from cattle liver abscesses than the subsp. funduliforme. Virulence factors and pathogenic mechanisms contributing to the formation of liver abscesses by F. necrophorum are poorly understood (Tan et al., 1996). However, several studies implicate leukotoxin to be a major virulence factor (Emery, et al., (1986) Generation of immunity against Fusobacterium necrophorum in mice inoculated with extracts containing leukotoxin. Vet. Microbiol. 12:255-268; Tan et al., 1996). The importance of leukotoxin is evidenced by correlation between toxin production and ability to induce abscesses in laboratory animals (Coyle-Dennis, et al., (1979) Correlation between leukocidin production and virulence of two isolates of Fusobacterium necrophorum. Am. J. Vet. Res. 40:274-276; Emery and Vaughn, 1986), inability of nonleukotoxin-producing strains to induce foot abscesses in cattle following intradermal inoculation (Emery, et al., (1985) Culture characteristics and virulence of strains of Fusobacterium necrophorum isolated from feet of cattle and sheep. Australian Vet. J. 62:43-46) and relationship between antileukotoxin antibody titers and protection in experimental challenge studies (Saginala, et al., (1996a) The serum neutralizing antibody response in cattle to Fusobacterium necrophorum leukotoxoid and possible protection against experimentally induced hepatic abscesses. Vet. Res. Comm., 20:493-504; Saginala, et al., (1996b) The serum neutralizing antibody response and protection against experimentally induced liver abscesses in steers vaccinated with Fusobacterium necrophorum. Am. J. Vet Res., 57:483-488; and Shinjo, et al., (1991) Proposal of two subspecies of Fusobacterium necrophorum (Flugge) Moore and Holdeman: Fusobacterium necrophorum subsp. necrophorum subsp. nov., nom. rev. (ex Flugge 1886), and Fusobacterium necrophorum subsp. funduliforme subsp. nov., nom. rev. (ex Hall 1898). Int. J. Sys. Bacteriol. 41:395-397).
Several investigators have attempted to induce protective immunity against F. necrophorum by using a variety of antigenic components. The results of such attempts have varied from ineffectual to significant protection. Clark et al. reported that cattle injected with F. necrophorum culture supernatant containing leukotoxin had a low incidence of foot rot caused by F. necrophorum. (Clark, et al. (1986), Studies into immunization of cattle against interdigital necrobacillosis. Aust. Vet. J. 63:107-110) Cell-free culture supernatant of a high leukotoxin producing strain of F. necrophorum (Tan et al., (1992) Factors affecting leukotoxin activity of F. necrophorum. Vet. Microbiol. 33:15-28), mixed with an adjuvant, was shown to elicit a high antileukotoxin antibody titer when injected in steers and provided significant protection to experimentally induced liver abscesses (Saginala et al., 1996a, b; 1997). F. necrophorum bacterin was used as an agent for immunizing cattle and sheep against liver necrosis as shown in EPO Application No. 460480 of Dec. 11, 1991 (the teachings of which are incorporated herein by reference). Specifically, virulent F. necrophorum isolates are inactivated using β-propiolactone, followed by addition of adjuvants. In addition, Abe et al., Infection and Immunity, 13:1473-1478, 1976 grew F. necrophorum for 48 hours. Cells were obtained by centrifuging, washing three times with saline, and were inactivated with formalin (0.4% in saline). The inactivated cells were then injected into mice to induce immunity. Two weeks after the last booster injection, each mouse was challenged with viable cells of F. necrophorum. The mice immunized with killed cells and challenged with live cells had no detectable bacteria in the liver, lung or spleen for up to 28 days. It was concluded that immunization of mice with formalin-killed F. necrophorum conferred protection against infection. Garcia et al. (Canadian J. Comp. Med, 38:222-226, 1974), conducted field trials to evaluate the efficacy of alum-precipitated toxoids of F. necrophorum. The vaccine preparation consisted of washed cells (unlikely to contain leukotoxin) that were ruptured by sonication. The most promising result was achieved with the injection of 15.5 mg protein of cytoplasmic toxoid. In this group, the incidents of liver abscesses was reduced to 10% from an average 35% in the control group. Emery et al., Vet. Microbiol., 12:255-268, 1986, prepared material by gel filtration of 18-hour culture supernate of F. necrophorum. This elicited significant immunity against challenge by with viable F. necrophorum. The injected preparation contained endotoxin and the majority of the leukotoxic activity. U.S. Pat. No. 5,455,034 (the teachings of which are incorporated herein by reference) by Nagaraja et al. disclosed that prevention of leukotoxin production (or inhibition of its activity) in immunized animals prevents the establishment of F. necrophorum infection. Thus, immunization of the animals against F. necrophorum leukotoxin, so that the animals' white blood cells or tissue macrophages may phagocytize the bacteria, presented a way to prevent diseases associated with F. necrophorum infection, e.g., liver abscesses in cattle and sheep, and foot rot in cattle. In order to produce such a leukotoxoid vaccine, the F. necrophorum bacteria was cultured in a way to enhance the elaboration of leukotoxin in the supernate. Thereupon, bacterial growth and leukotoxin elaboration was terminated, and a vaccine prepared by inactivating at least the leukotoxin-containing supernate. In more detail, the leukotoxin elaboration method of the '034 patent involved first forming a culture of F. necrophorum bacteria in growth media, and thereafter causing the bacteria to grow in the culture and to simultaneously elaborate leukotoxin in the supernate. At the end of the culturing step, i.e., at the end of the selected culture time within the range of from about 4-10 hours, the bacterial growth and leukotoxin elaboration were terminated, and the leukotoxoid vaccine was prepared. This involved first separating the leukotoxin-containing supernate from the bacteria, followed by inactivation through use of formalin, β-propiolactone, heat, radiation or any other known method of inactivation. Alternately, the entire culture could be inactivated to form the vaccine.
Presently, the control of liver abscesses is with the use of antimicrobial feed additives. Antimicrobial compounds reduce the incidence of liver abscesses but do not eliminate the problem (Nagaraja et al., 1998). Therefore, an effective vaccine would be highly desirable to the feedlot industry. The vaccine approach also would alleviate public health concerns associated with the use of subtherapeutic levels of antibiotics in the feed. Because studies have indicated that antileukotoxin immunity reduces the incidence of hepatic abscesses and interdigital necrobacillosis (Garcia et al., 1974; Clark et al., 1986; Saginala et al., 1996a, b; 1997), the development of a recombinant leukotoxin vaccine will be of great value in the control of hepatic and interdigital necrobacillosis in cattle.